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Differential roles of ArfGAP1, ArfGAP2, and ArfGAP3 in COPI trafficking
Correspondence to F. Wieland: felix.wieland{at}bzh.uni-heidelberg.de
The formation of coat protein complex I (COPI)–coated vesicles is regulated by the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor 1 (Arf1), which in its GTP-bound form recruits coatomer to the Golgi membrane. Arf GTPase-activating protein (GAP) catalyzed GTP hydrolysis in Arf1 triggers uncoating and is required for uptake of cargo molecules into vesicles. Three mammalian ArfGAPs are involved in COPI vesicle trafficking; however, their individual functions remain obscure. ArfGAP1 binds to membranes depending on their curvature. In this study, we show that ArfGAP2 and ArfGAP3 do not bind directly to membranes but are recruited via interactions with coatomer. In the presence of coatomer, ArfGAP2 and ArfGAP3 activities are comparable with or even higher than ArfGAP1 activity. Although previously speculated, our results now demonstrate a function for coatomer in ArfGAP-catalyzed GTP hydrolysis by Arf1. We suggest that ArfGAP2 and ArfGAP3 are coat protein–dependent ArfGAPs, whereas ArfGAP1 has a more general function.
© 2008 Weimer et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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Introduction |
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The ArfGAP family of cytosolic proteins is characterized by a well-conserved catalytical zinc finger domain, whereas their noncatalytical domains differ between subgroups of the family (Randazzo and Hirsch, 2004). Two ArfGAPs have been implicated in COPI transport in yeast, Gcs1 and Glo3 (Poon et al., 1999). Both proteins provide overlapping functions and can restore single knockouts of the respective other ArfGAP, but a double knockout of Gcs1 and Glo3 is lethal. The mammalian homologue of Gcs1, ArfGAP1, was the first ArfGAP to be identified (Cukierman et al., 1995; Makler et al., 1995), and its role in COPI trafficking has been studied intensively (Huber et al., 1998; Goldberg, 1999; Bigay et al., 2003; Liu et al., 2005). ArfGAP2 and ArfGAP3, both mammalian homologues of Glo3, have been shown only recently to be involved in COPI vesicle trafficking (Frigerio et al., 2007). Consistent with the findings in yeast, triple knockdowns in mammalian cells are lethal, whereas cells can survive when only ArfGAP1 or both ArfGAP2 and ArfGAP3 are silenced.
ArfGAP1, ArfGAP2, and ArfGAP3 show high sequence similarity within the very N-terminal catalytical domain. In ArfGAP1, two ArfGAP1 lipid packing sensory (ALPS) motifs have been identified within the noncatalytical domain (Bigay et al., 2005; Mesmin et al., 2007). ALPS motifs are unstructured in solution but form an amphipathic 
helix once bound to highly curved membranes as present on a vesicle. Because of this binding behavior, ArfGAP1 displays curvature-dependent ArfGAP activity in vitro, a mechanism suggested to ensure high uncoating efficiency on vesicles, whereas basal activity on flat membranes is rather low (Bigay et al., 2003, 2005). The noncatalytical domains of ArfGAP2 and ArfGAP3 differ from that of ArfGAP1 and show 50% overall sequence identity (Frigerio et al., 2007). There is evidence for an essential functional role of a highly conserved C-terminal motif, the Glo3 motif, which has not been further characterized (Yahara et al., 2006). A recent study revealed that the noncatalytical domains of ArfGAP2 and ArfGAP3 interact with coatomer (Frigerio et al., 2007).
A role of coatomer in ArfGAP-mediated GTP hydrolysis has been studied in different systems. A 100–1,000-fold stimulatory effect of coatomer on GTP hydrolysis was described for the catalytical domain of ArfGAP1 when a soluble version of Arf1, N
17Arf1, was used (Goldberg, 1999). However, only very weak (less than twofold) stimulation by coatomer of full-length ArfGAP1 was found in an assay using full-length myristoylated Arf1 on Golgi membranes. In contrast, the activity of Glo3 was increased significantly (
50-fold) in the presence of coatomer (Szafer et al., 2001).
Previous work on ArfGAP activities in COPI vesicle trafficking does not explain functionally the existence of several ArfGAPs (Huber et al., 1998; Yang et al., 2002; Liu et al., 2005; Frigerio et al., 2007). Therefore, the purpose of this study was to characterize the three mammalian ArfGAPs involved in COPI vesicle trafficking with respect to their individual mechanisms. To this end, the recombinant proteins were expressed in insect cells, purified, and characterized. We find that, in contrast to ArfGAP1, ArfGAP2 and ArfGAP3 activities are dependent on coatomer. From our data, we conclude that coatomer is required for efficient recruitment of ArfGAP2 and ArfGAP3 to the Golgi membrane. Once recruited by coatomer, ArfGAP2 and ArfGAP3 exhibit a strikingly higher activity than ArfGAP1. Two different mechanisms regulate the activities of the three ArfGAPs: binding to membranes by ALPS motifs in the case of ArfGAP1 and recruitment via binding to coatomer in the case of ArfGAP2 and ArfGAP3. These two mechanisms implicate different roles of the three ArfGAPs: ArfGAP2 and ArfGAP3 are the coat protein–dependent ArfGAPs, whereas ArfGAP1 has COPI-dependent as well as COPI-independent functions.
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ArfGAP2 and ArfGAP3 display weak and curvature-independent activity in a liposome-based GAP assay
ArfGAP1 and its yeast homologue Gcs1 were reported to catalyze GTP hydrolysis in Arf1 in a liposome-based fluorimetric assay (Bigay and Antonny, 2005). GAP activity was observed to be increased on highly curved membranes because of increased binding of the ALPS motif to the liposomes. Therefore, we compared the activities of ArfGAP1 with ArfGAP2 and ArfGAP3 in the same assay. GAP-catalyzed GTP hydrolysis in Arf1 was measured as described by Bigay et al. (2005) on liposomes of 200- or 30-nm diameter in a spectrofluorometer. The exchange of GDP for GTP on myristoylated full-length Arf1 is measured as an increase in tryptophan fluorescence. After the addition of ArfGAPs, fluorescence decreases because of GTP hydrolysis (Fig. 3).
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ArfGAP2 and ArfGAP3 fail to bind to liposomes
To address the question of whether the weaker activities of ArfGAP2 and ArfGAP3 compared with ArfGAP1 correlate with a different binding behavior to liposomes, we performed flotation assays on sucrose density gradients (Bigay and Antonny, 2005). The fractions containing liposomes were analyzed for bound proteins by Western blotting with specific peptide antibodies raised against each of the ArfGAPs.
Curvature-dependent binding of ArfGAP1 to the membranes as shown by Bigay et al. (2005) was reproduced. As shown in Fig. 4 A, 10% of the input is bound to the largest liposomes (200 nm), and binding is increased fivefold when the liposomal diameter was reduced to 30 nm, which is consistent with published data.
In comparison, ArfGAP2 and ArfGAP3 fail to bind to liposomes under these experimental conditions.
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ArfGAP1, ArfGAP2, and ArfGAP3 are recruited to the Golgi membrane by different mechanisms
According to immunofluorescence experiments (Cukierman et al., 1995; Frigerio et al., 2007) and immunogold-labeling experiments (Fig. 2 A), all three ArfGAPs are found to be located to Golgi membranes. Therefore, we next addressed the question by which mechanism ArfGAP2 and ArfGAP3 are recruited to the membrane if not by direct binding to the lipid bilayer. A promising candidate to mediate this binding was coatomer, the COP of COPI vesicles, because interaction with coatomer of ArfGAP2 and ArfGAP3 was described previously (Frigerio et al., 2007). To investigate this possibility, we incubated Golgi membranes with ArfGAPs in the presence or absence of the coat proteins coatomer and Arf1. After centrifugation, the pelleted membranes were analyzed for bound proteins (Fig. 4 B).
We observed binding of 20–30% of the provided ArfGAP1 to Golgi membranes (Fig. 4 B, lane 9). The amount of bound protein is not affected by the presence of Arf1-GTP
S (Fig. 4 B, lane 10), and only a small increase, by less than a factor of two, is found when coatomer was provided additionally (Fig. 4 B, lane 11). In contrast, almost no ArfGAP2 and ArfGAP3 were detected on the Golgi membranes in the presence or absence of Arf1-GTPS (Fig. 4 B, lanes 9 and 10). However, binding is significantly increased when coatomer is bound to the Golgi (Fig. 4 B, lane 11). These results are consistent with previous findings that ArfGAP2 and ArfGAP3 bind to coatomer, as shown in pull-down assays (Watson et al., 2004; Frigerio et al., 2007).
In summary, these results suggest that the three ArfGAPs are recruited to the Golgi membrane in comparable amounts but by different mechanisms. ArfGAP1 inserts directly into the membrane via its ALPS domains, preferentially at sites with high curvature, whereas ArfGAP2 and ArfGAP3 bind to coatomer and thereby are recruited indirectly to the membrane.
The three ArfGAPs antagonize the formation of coated vesicles in a COPI-budding assay
To test the effect of ArfGAPs on COPI-coated vesicle formation, we performed COPI vesicle–budding assays using Golgi membranes and recombinant proteins under GTP conditions. Each ArfGAP was titrated into the budding reaction, and COPI-coated vesicles were purified by sucrose density centrifugation defined by their buoyant density (see Materials and methods). Fractions were analyzed for vesicle yield as determined by Western blot signals for the transmembrane protein p23 and the coat proteins Arf1 and coatomer. The signals were normalized to an experiment without added ArfGAPs, as depicted in Fig. 5 A.
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As the assay was performed with Golgi membranes in the presence of coatomer, all three ArfGAPs should be recruited to the membrane (Fig. 4 B), where they can act on their substrate Arf1-GTP. These results suggest a similar GAP activity of ArfGAP1, ArfGAP2, and ArfGAP3 either caused by direct membrane binding or recruitment by coatomer. However, with this assay it is not possible to tell apart inhibition of COPI-coated vesicle formation (caused by rapid release of Arf1 from the membrane) from rapid uncoating of COPI-coated vesicles.
Collectively, these data show that the three ArfGAPs antagonize the yield of COPI-coated vesicles to roughly the same extent. To more precisely quantify uncoating activities, static light–scattering assays with COPI-coated liposomes as well as Golgi membranes were performed.
ArfGAP2 and ArfGAP3 are more efficient in uncoating than ArfGAP1
ArfGAP-catalyzed GTP hydrolysis in Arf1 leads to uncoating of COPI vesicles (Tanigawa et al., 1993; Cukierman et al., 1995; Reinhard et al., 2003). To assess the activities of the three ArfGAPs in this process, we used a light-scattering assay as described previously (Bigay and Antonny, 2005) that allows observance of the assembly–disassembly cycle of the COPI coat. To this end, liposomes containing p23 lipopeptide (Nickel and Wieland, 2001) are incubated with Arf1, coatomer, and EDTA. The coating reaction is started by the addition of GTP and is followed by a proportional increase in light scattering. After reaching a plateau, the GTP state of Arf1 was stabilized by the addition of Mg2+, and uncoating of the liposomes was initiated by the addition of the individual ArfGAPs.
To control for the requirement of GTP hydrolysis in the uncoating reaction, liposomes were analyzed that were coated in the presence of the poorly hydrolyzable GTP analogue GTPS. As shown in Fig. 6 B, uncoating after the addition of ArfGAPs was prevented, linking the uncoating reaction to the activity of the ArfGAPs.
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To investigate the ArfGAP activities under more physiological conditions, static light–scattering experiments were performed with Golgi-enriched membranes. As depicted in Fig. 6 D, on Golgi membranes the three ArfGAPs qualitatively showed activities similar to those on liposomes.
Quantification of initial reaction rates showed ratios of ArfGAP1 to ArfGAP2/ArfGAP3 activities of 1:10 for 160-nm liposomes, 1:5 for 60- or 40-nm liposomes, and 1:3 for Golgi membranes. This may either reflect the presence of additional ArfGAP effectors on the Golgi or preferential coating at sites with high curvature. In summary, when analyzed in the COPI-budding assay, the three ArfGAPs showed comparable activities, whereas in the uncoating assay, the activities of ArfGAP2 and ArfGAP3 are markedly higher.
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Collectively, we find that regulation of ArfGAP2 and ArfGAP3 activities is based on a mechanism distinct from ArfGAP1, and our results suggest that the roles of the Glo3 homologues in COPI trafficking differ from those of ArfGAP1.
A role of coatomer in ArfGAP-mediated GTP hydrolysis
An effect of coatomer on GTP hydrolysis catalyzed by ArfGAPs has been investigated before, yet its precise role is not clear. Goldberg (1999) analyzed a truncated soluble form of Arf1, N17Arf1, with the catalytical domain of ArfGAP1 in the absence of membranes. In this assay, ArfGAP1 catalytical domain is present at very high concentrations almost stoichiometric to Arf1, suggesting a very low activity of ArfGAP1 in solution. Addition of coatomer to this system results in a strong (up to 1,000-fold) stimulation of GTP hydrolysis, indicating that in solution, Arf1-GTP, when bound to coatomer, represents a better substrate for the ArfGAP. In contrast, Szafer et al. (2000) investigated ArfGAP1 activity in a liposomal system with the full-length protein and myristoylated full-length Arf1. In this assay, GTP hydrolysis activity is observed at a much lower concentration of ArfGAP, in the nanomolar range. This high activity is not further stimulated by the addition of coatomer. This is consistent with an ALPS-dependent binding to membranes of ArfGAP1 (Bigay et al., 2003, 2005) that is not expected to be further enhanced by coatomer. Likewise, on Golgi membranes, only a very weak stimulation of ArfGAP1 activity by coatomer was observed. In contrast, Glo3 activity is highly stimulated by coatomer on Golgi membranes but only weakly on liposomes (Szafer et al., 2001). In light of the data presented in this study, this lack of stimulation on liposomes could be explained by the absence of p24 tails that are required for efficient recruitment of coatomer (Bremser et al., 1999) and thereby of Glo3 to the membrane.
A direct interaction of the yeast Glo3 but not Gcs1 with the coatomer complex has been published (Eugster et al., 2000; Lewis et al., 2004). Moreover, the noncatalytical domains of both Glo3 and ArfGAP2 were found to bind coatomer in almost stoichiometric amounts, as shown by pull-down assays (Frigerio et al., 2007). Collectively, our data explain the role of coatomer in ArfGAP-catalyzed GTP hydrolysis. It seems that coatomer does not provide a catalytical cofactor activity but simply acts as a binding partner of Glo3 homologues for efficient recruitment to the membrane. ArfGAP1 acts independently of coatomer, as it binds to the lipid bilayer by its ALPS domains.
Roles of ArfGAPs in COPI trafficking
The three ArfGAPs are likely to have overlapping functions and are probably able to substitute for each other, as can be deduced from knockdown experiments in yeast and human cells (Poon et al., 1999; Frigerio et al., 2007). However, the two differential modes of membrane binding, coatomer independent in the case of ArfGAP1 and coatomer dependent in the case of ArfGAP2 and ArfGAP3, implicate different roles of the three enzymes. From our data, we conclude that ArfGAP2 and ArfGAP3 are coat protein–dependent ArfGAPs, whereas ArfGAP1 plays a more general role, as its activity does not depend on coatomer. ArfGAP1 has been shown to be involved in COPI-dependent as well as COPI-independent events, such as a regulation of an asymmetrical mode of tethering between flat and curved membranes of the golgin GMAP-210 (Drin et al., 2008), further supporting differential functions of the three ArfGAPs. The strictly coatomer-dependent activities of ArfGAP2 and ArfGAP3 are reminiscent of the mechanism of coat-controlled specificity of GTP hydrolysis that is also found for the COPII and clathrin coats. The Sar1 GAP activity is mediated by a subunit of the COPII coat itself, Sec23 (Yoshihisa et al., 1993; Bi et al., 2002). In clathrin-dependent trafficking, direct specific interactions of the ArfGAPs AGAP1 and AGAP2 with the adapter protein complexes AP-1 and AP-3, respectively, have been shown, ensuring site-specific regulation (Nie et al., 2003, 2005). Whether ArfGAP2 or ArfGAP3 is bound to coatomer already in the cytosol and recruited en bloc with the complex or whether coatomer is bound first and recruits the ArfGAPs in a second step cannot be distinguished at this time. However, the light-scattering data show that ArfGAP2 and ArfGAP3 can be recruited after coatomer has attached to the membrane, and their specific activities in this uncoating assay are markedly higher than that of ArfGAP1. Individual roles of ArfGAP2 and ArfGAP3 will be the subject of future work. One could imagine that the two proteins interact differentially with coatomer isoforms (Futatsumori et al., 2000; Wegmann et al., 2004; Moelleken et al., 2007) and therefore might discriminate between different subpopulations of COPI vesicles comparable with AGAP1 and AGAP2 that distinguish between the adapter protein complexes AP-1 and AP-3 (Nie et al., 2005). Thus, once coatomer isoforms are available, questions as to the functional differences of ArfGAP2 and ArfGAP3 can be addressed directly.
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Materials and Methods |
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Generation of ArfGAP-specific antibodies
Peptide antibodies specific for ArfGAP1 (GAP1 rab2), ArfGAP2 (GAP2 rab2), and ArfGAP3 (GAP3 rab2) were raised in rabbits (Pineda Antikörper Service) using the following peptides: EPPKAKSPSSDSWTC (ArfGAP1), CTFASGPPKYKDNPFS (ArfGAP2), and CKYQEDPEDSYFSSSSK (ArfGAP3). Antisera were affinity purified and tested for specificity in Western blotting, immunoprecipitation, and immunofluorescence.
Preparation of Arf1, coatomer, and Golgi membranes
Full-length myristoylated human Arf1 was recombinantly expressed and purified as described previously (Franco et al., 1995). Rabbit liver coatomer was purified as described previously (Pavel et al., 1998). Golgi membranes were enriched from rat liver as described previously (Tabas and Kornfeld, 1979).
Liposome preparation
Liposomes of a Golgi-like lipid composition ± p23 lipopeptide (Nickel and Wieland, 2001) were generated as described previously (Bigay et al., 2005) and extruded through polycarbonate filters of 200-, 80-, or 30-nm pore size (Avestin or GE Healthcare). To determine the actual diameter, aliquots of the liposomes were diluted to a final concentration of 0.1 mM in 50 µl of HKM buffer (25 mM Hepes-KOH, 150 mM KCl, and 1 mM MgCl2, pH 7.4) and measured at RT in a Zetasizer instrument (1000HS; Malvern). Data were acquired using the Zetasizer advanced software (1000HS). The measured mean diameters were approximately 160 nm, 60 nm, and 40 nm.
Tryptophan fluorescence assay
ArfGAP activity on liposomes was measured as described previously (Bigay et al., 2005) in a spectrofluorometer (FP-6500; Jasco) at 37°C in HKM buffer in a total volume of 600 µl using 100 µM of liposomes and 1 µM Arf1. GTP exchange was started by the addition of 100 µM GTP and 2 mM EDTA. After 750 s, MgCl2 was added to a final concentration of 4 mM followed by the addition of ArfGAPs to a final concentration of 50 nM, 500 nM, or 1 µM. Fluorescence was normalized to zero before the addition of EDTA (GDP state) and to 100 AU after the addition of MgCl2 (GTP state). The shift in fluorescence caused by the addition of ArfGAPs was subtracted.
As controls, ArfGAP proteins alone (final concentrations of 50 nM, 500 nM, or 1 µM) were added to HKM buffer. Tryptophan fluorescence was measured using the aforementioned parameters. Fluorescence levels at the time points of ArfGAP addition were normalized to 100 AU.
Membrane-binding assays
Flotation experiments with liposomes were performed in HKM buffer as described previously (Bigay and Antonny, 2005) with the following variations. Instead of NBD-labeled lipid, rhodamine-labeled phosphatidylethanolamine (Avanti Polar Lipids, Inc.) was used. After SDS-PAGE, bound proteins were stained by Western blotting with ArfGAP-specific antibodies.
Golgi-binding assays were performed in assay buffer (25 mM Hepes-KOH, 20 mM KCl, and 2.5 mM MgAc, pH 7.2) using the following components: 100 µg of Golgi membranes, 5 µg Arf1, 32 µg coatomer, 0.1 mM GTPS, and 1 µg of each ArfGAP in a total volume of 200 µl. After incubation for 5 min at RT, the samples were layered on a 300-µl cushion of 20% (vol/vol) sucrose. Golgi membranes were pelleted by centrifugation at 15,000 g for 20 min at 4°C in a rotor (SW55Ti; Beckman Coulter), resuspended in sample buffer, and analyzed for bound proteins by SDS-PAGE and Western blotting.
Vesicle-budding assay
To reconstitute COPI vesicles, 0.8 mg of salt-washed Golgi membranes was mixed with 5 µg Arf1, 30 µg coatomer, and 1.2 mM GTP in a total volume of 250 µl. ArfGAPs were titrated in from 0.025–25 pmol. After 10-min incubation at 37°C, the salt concentration was raised to 250 mM KCl. After centrifugation at 12,000 g for 5 min, the supernatant containing COPI vesicles was loaded on top of two sucrose cushions (10 µl of 50% sucrose and 50 µl of 37% sucrose) and centrifuged at 100,000 g for 50 min in an SW55Ti rotor. COPI-coated vesicles were concentrated at the interphase between 50 and 37% sucrose. 0.5% of the input sample and 50% of the isolated vesicle fraction were analyzed by Western blotting using an Alexa Fluor 680–conjugated goat anti–rabbit secondary antibody (Invitrogen). Signals were quantified using an infrared imaging system and software (Odyssey; LI-COR Biosciences).
Light-scattering assay
Light-scattering assays were performed as described previously (Bigay and Antonny, 2005) using 70 µg/ml of salt-washed Golgi membranes or 0.1 mM Golgi-like liposomes supplemented with 2 mol% p23 lipopeptide, 1 µM Arf1, 2 mM EDTA, and 100 µM GTP or GTPS in static light–scattering buffer (50 mM Hepes-KOH, pH 7.2, 1 mM MgCl2, 1 mM DTT, and 120 mM KAc) in a total volume of 600 µl. After 10-min incubation at 37°C, 4 mM MgCl2 was added followed by the addition of recombinant ArfGAPs to a final concentration of 1, 2.5, or 5 nM. Scattering was normalized to 0 AU before the addition of GTP and to 100 AU before the addition of MgCl2.
Quantification
NRK cells grown to 80% confluency were trypsinized and suspended in PBS. Cells were pelleted (1 min at 1,000 g), washed once with PBS and once with breaking buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.8 µg/ml pepstatin A, 1 mM o-phenanthrolin, and protease inhibitor cocktail [Roche]), resuspended in breaking buffer, and lysed by several passages through 24- and 27-G needles until >90% were broken. Supernatants of a 1,000 g centrifugation were analyzed by Western blotting. Known amounts of purified proteins were loaded as standards. Western blot signals were quantified using the Odyssey infrared imaging system.
Immunogold labeling
Immunogold-labeling experiments were performed as described previously (Moelleken et al., 2007). In brief, cryosections of chemically fixed NRK cells were labeled with an ArfGAP-specific antibody followed by incubation with protein A–coupled gold particles of 15-nm size (provided by G. Posthuma, Utrecht University, Netherlands). GM130 antibody (Abcam) was used as a cis-Golgi marker and visualized with 10-nm gold particles conjugated to either protein A or goat anti–mouse antibody (British BioCell International). The sections were contrasted with uranyl acetate and analyzed by transmission electron microscopy (EM 10 CR; Carl Zeiss, Inc.). Digital images were acquired with a 300-s exposure time using a wide angle dual speed SlowScan charge-coupled device camera (WIA-7888-V; Tröndle Restlichtverstärkersysteme) for transmission electron microscopy and the corresponding ImageSP software (Tröndle Restlichtverstärkersysteme). Gold dots of 15 nm representing ArfGAPs were counted, and the relative labeling density at cis- and trans-Golgi was calculated.
Online supplemental material
Fig. S1 shows the kinetics of tryptophan fluorescence of ArfGAP1, ArfGAP2, and ArfGAP3 as a control for the ArfGAP activity assays. Fig. S2 shows the actual diameter of liposomes as measured by dynamic light scattering. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200806140/DC1.
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Acknowledgments |
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Submitted: 23 June 2008
Accepted: 21 October 2008
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References |
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Antonny, B., S. Beraud-Dufour, P. Chardin, and M. Chabre. 1997. N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange. Biochemistry. 36:4675–4684.[CrossRef][Medline]
Beck, R., Z. Sun, F. Adolf, C. Rutz, J. Bassler, K. Wild, I. Sinning, E. Hurt, B. Brugger, J. Bethune, and F. Wieland. 2008. Membrane curvature induced by Arf1-GTP is essential for vesicle formation. Proc. Natl. Acad. Sci. USA. 105:11731–11736.
Bethune, J., F. Wieland, and J. Moelleken. 2006. COPI-mediated Transport. J. Membr. Biol. 211:65–79.[CrossRef][Medline]
Bi, X., R.A. Corpina, and J. Goldberg. 2002. Structure of the Sec23/24-Sar1 pre-budding complex of the COPII vesicle coat. Nature. 419:271–277.[CrossRef][Medline]
Bigay, J., and B. Antonny. 2005. Real-time assays for the assembly-disassembly cycle of COP coats on liposomes of defined size. Methods Enzymol. 404:95–107.[CrossRef][Medline]
Bigay, J., P. Gounon, S. Robineau, and B. Antonny. 2003. Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature. Nature. 426:563–566.[CrossRef][Medline]
Bigay, J., J.F. Casella, G. Drin, B. Mesmin, and B. Antonny. 2005. ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif. EMBO J. 24:2244–2253.[CrossRef][Medline]
Bremser, M., W. Nickel, M. Schweikert, M. Ravazzola, M. Amherdt, C.A. Hughes, T.H. Sollner, J.E. Rothman, and F.T. Wieland. 1999. Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors. Cell. 96:495–506.[CrossRef][Medline]
Cukierman, E., I. Huber, M. Rotman, and D. Cassel. 1995. The ARF1 GTPase-activating protein: zinc finger motif and Golgi complex localization. Science. 270:1999–2002.
Dogic, D., B. de Chassey, E. Pick, D. Cassel, Y. Lefkir, S. Hennecke, P. Cosson, and F. Letourneur. 1999. The ADP-ribosylation factor GTPase-activating protein Glo3p is involved in ER retrieval. Eur. J. Cell Biol. 78:305–310.[Medline]
Drin, G., V. Morello, J.F. Casella, P. Gounon, and B. Antonny. 2008. Asymmetric tethering of flat and curved lipid membranes by a golgin. Science. 320:670–673.
Eugster, A., G. Frigerio, M. Dale, and R. Duden. 2000. COP I domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP. EMBO J. 19:3905–3917.[CrossRef][Medline]
Franco, M., P. Chardin, M. Chabre, and S. Paris. 1995. Myristoylation of ADP-ribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels. J. Biol. Chem. 270:1337–1341.
Franco, M., P. Chardin, M. Chabre, and S. Paris. 1996. Myristoylation-facilitated binding of the G protein ARF1GDP to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor. J. Biol. Chem. 271:1573–1578.
Frigerio, G., N. Grimsey, M. Dale, I. Majoul, and R. Duden. 2007. Two human ARFGAPs associated with COP-I-coated vesicles. Traffic. 8:1644–1655.[CrossRef][Medline]
Futatsumori, M., K. Kasai, H. Takatsu, H.W. Shin, and K. Nakayama. 2000. Identification and characterization of novel isoforms of COP I subunits. J. Biochem. 128:793–801.
Goldberg, J. 1999. Structural and functional analysis of the ARF1-ARFGAP complex reveals a role for coatomer in GTP hydrolysis. Cell. 96:893–902.[CrossRef][Medline]
Gommel, D.U., A.R. Memon, A. Heiss, F. Lottspeich, J. Pfannstiel, J. Lechner, C. Reinhard, J.B. Helms, W. Nickel, and F.T. Wieland. 2001. Recruitment to Golgi membranes of ADP-ribosylation factor 1 is mediated by the cytoplasmic domain of p23. EMBO J. 20:6751–6760.[CrossRef][Medline]
Honda, A., O.S. Al-Awar, J.C. Hay, and J.G. Donaldson. 2005. Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE. J. Cell Biol. 168:1039–1051.
Huber, I., E. Cukierman, M. Rotman, T. Aoe, V.W. Hsu, and D. Cassel. 1998. Requirement for both the amino-terminal catalytic domain and a noncatalytic domain for in vivo activity of ADP-ribosylation factor GTPase-activating protein. J. Biol. Chem. 273:24786–24791.
Huber, I., M. Rotman, E. Pick, V. Makler, L. Rothem, E. Cukierman, and D. Cassel. 2001. Expression, purification, and properties of ADP-ribosylation factor (ARF) GTPase activating protein-1. Methods Enzymol. 329:307–316.[CrossRef][Medline]
Lanoix, J., J. Ouwendijk, A. Stark, E. Szafer, D. Cassel, K. Dejgaard, M. Weiss, and T. Nilsson. 2001. Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1. J. Cell Biol. 155:1199–1212.
Lewis, S.M., P.P. Poon, R.A. Singer, G.C. Johnston, and A. Spang. 2004. The ArfGAP Glo3 is required for the generation of COPI vesicles. Mol. Biol. Cell. 15:4064–4072.
Liu, W., R. Duden, R.D. Phair, and J. Lippincott-Schwartz. 2005. ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells. J. Cell Biol. 168:1053–1063.
Liu, X., C. Zhang, G. Xing, Q. Chen, and F. He. 2001. Functional characterization of novel human ARFGAP3. FEBS Lett. 490:79–83.[CrossRef][Medline]
Makler, V., E. Cukierman, M. Rotman, A. Admon, and D. Cassel. 1995. ADP-ribosylation factor-directed GTPase-activating protein. Purification and partial characterization. J. Biol. Chem. 270:5232–5237.
Malsam, J., D. Gommel, F.T. Wieland, and W. Nickel. 1999. A role for ADP ribosylation factor in the control of cargo uptake during COPI-coated vesicle biogenesis. FEBS Lett. 462:267–272.[CrossRef][Medline]
Mesmin, B., G. Drin, S. Levi, M. Rawet, D. Cassel, J. Bigay, and B. Antonny. 2007. Two lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to membrane curvature. Biochemistry. 46:1779–1790.[CrossRef][Medline]
Moelleken, J., J. Malsam, M.J. Betts, A. Movafeghi, I. Reckmann, I. Meissner, A. Hellwig, R.B. Russell, T. Sollner, B. Brugger, and F.T. Wieland. 2007. Differential localization of coatomer complex isoforms within the Golgi apparatus. Proc. Natl. Acad. Sci. USA. 104:4425–4430.
Nickel, W., and F.T. Wieland. 2001. Receptor-dependent formation of COPI-coated vesicles from chemically defined donor liposomes. Methods Enzymol. 329:388–404.[CrossRef][Medline]
Nickel, W., J. Malsam, K. Gorgas, M. Ravazzola, N. Jenne, J.B. Helms, and F.T. Wieland. 1998. Uptake by COPI-coated vesicles of both anterograde and retrograde cargo is inhibited by GTPgammaS in vitro. J. Cell Sci. 111:3081–3090.[Abstract]
Nie, Z., M. Boehm, E.S. Boja, W.C. Vass, J.S. Bonifacino, H.M. Fales, and P.A. Randazzo. 2003. Specific regulation of the adaptor protein complex AP-3 by the Arf GAP AGAP1. Dev. Cell. 5:513–521.[CrossRef][Medline]
Nie, Z., J. Fei, R.T. Premont, and P.A. Randazzo. 2005. The Arf GAPs AGAP1 and AGAP2 distinguish between the adaptor protein complexes AP-1 and AP-3. J. Cell Sci. 118:3555–3566.
Palmer, D.J., J.B. Helms, C.J. Beckers, L. Orci, and J.E. Rothman. 1993. Binding of coatomer to Golgi membranes requires ADP-ribosylation factor. J. Biol. Chem. 268:12083–12089.
Pavel, J., C. Harter, and F.T. Wieland. 1998. Reversible dissociation of coatomer: functional characterization of a beta/delta-coat protein subcomplex. Proc. Natl. Acad. Sci. USA. 95:2140–2145.
Pepperkok, R., J.A. Whitney, M. Gomez, and T.E. Kreis. 2000. COPI vesicles accumulating in the presence of a GTP restricted arf1 mutant are depleted of anterograde and retrograde cargo. J. Cell Sci. 113:135–144.[Abstract]
Poon, P.P., D. Cassel, A. Spang, M. Rotman, E. Pick, R.A. Singer, and G.C. Johnston. 1999. Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function. EMBO J. 18:555–564.[CrossRef][Medline]
Randazzo, P.A., and D.S. Hirsch. 2004. Arf GAPs: multifunctional proteins that regulate membrane traffic and actin remodelling. Cell. Signal. 16:401–413.[CrossRef][Medline]
Reinhard, C., C. Harter, M. Bremser, B. Brugger, K. Sohn, J.B. Helms, and F. Wieland. 1999. Receptor-induced polymerization of coatomer. Proc. Natl. Acad. Sci. USA. 96:1224–1228.
Reinhard, C., M. Schweikert, F.T. Wieland, and W. Nickel. 2003. Functional reconstitution of COPI coat assembly and disassembly using chemically defined components. Proc. Natl. Acad. Sci. USA. 100:8253–8257.
Singh, J., Y. Itahana, S. Parrinello, K. Murata, and P.Y. Desprez. 2001. Molecular cloning and characterization of a zinc finger protein involved in Id-1-stimulated mammary epithelial cell growth. J. Biol. Chem. 276:11852–11858.
Szafer, E., E. Pick, M. Rotman, S. Zuck, I. Huber, and D. Cassel. 2000. Role of coatomer and phospholipids in GTPase-activating protein-dependent hydrolysis of GTP by ADP-ribosylation factor-1. J. Biol. Chem. 275:23615–23619.
Szafer, E., M. Rotman, and D. Cassel. 2001. Regulation of GTP hydrolysis on ADP-ribosylation factor-1 at the Golgi membrane. J. Biol. Chem. 276:47834–47839.
Tabas, I., and S. Kornfeld. 1979. Purification and characterization of a rat liver Golgi alpha-mannosidase capable of processing asparagine-linked oligosaccharides. J. Biol. Chem. 254:11655–11663.
Tanigawa, G., L. Orci, M. Amherdt, M. Ravazzola, J.B. Helms, and J.E. Rothman. 1993. Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi-derived COP-coated vesicles. J. Cell Biol. 123:1365–1371.
Watson, P.J., G. Frigerio, B.M. Collins, R. Duden, and D.J. Owen. 2004. Gamma-COP appendage domain - structure and function. Traffic. 5:79–88.[CrossRef][Medline]
Wegmann, D., P. Hess, C. Baier, F.T. Wieland, and C. Reinhard. 2004. Novel isotypic gamma/zeta subunits reveal three coatomer complexes in mammals. Mol. Cell. Biol. 24:1070–1080.
Yahara, N., K. Sato, and A. Nakano. 2006. The Arf1p GTPase-activating protein Glo3p executes its regulatory function through a conserved repeat motif at its C-terminus. J. Cell Sci. 119:2604–2612.
Yang, J.S., S.Y. Lee, M. Gao, S. Bourgoin, P.A. Randazzo, R.T. Premont, and V.W. Hsu. 2002. ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat. J. Cell Biol. 159:69–78.
Yoshihisa, T., C. Barlowe, and R. Schekman. 1993. Requirement for a GTPase-activating protein in vesicle budding from the endoplasmic reticulum. Science. 259:1466–1468.
Zhao, X., A. Claude, J. Chun, D.J. Shields, J.F. Presley, and P. Melancon. 2006. GBF1, a cis-Golgi and VTCs-localized ARF-GEF, is implicated in ER-to-Golgi protein traffic. J. Cell Sci. 119:3743–3753.
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-COP in Western blots. Signals were quantified on the basis of standards of the respective recombinant proteins. The diagram shows the amounts in picomoles per milligram of total protein in the lysates of seven independent experiments. (B and C) Error bars represent standard deviations of the mean.
-COP, Arf1, and p23. MW, molecular weight. (B) Western blot signals for p23, Arf1, and 